Acute diarrheal disease due to transient colonization of the small bowel by enterotoxigenic strains of Escherichia coli (E. coli or ETEC) is a major health problem of global scope for both humans and for animal husbandry. These organisms, together with rotavirus and Campylobacter jejuni (C. jejuni), are the principal cause of the often fatal acute diarrhea that is common among infants living in underdeveloped countries and among neonatal animals, particularly lambs and piglets. ETEC strains are also the usual cause of acute diarrhea among persons from temperate zones who travel to the tropics, and may be responsible for sporadic or epidemic episodes of diarrhea among children and adults living in either temperate or tropical areas.
The disease caused by ETEC is mediated by the release of two enterotoxins, either singly or together. The low molecular weight, heat-stable toxin (ST) produced by ETEC strains of human or porcine origin has recently been purified. Preparations of ST have a relatively high content of half-cystine, cause secretion by stimulating guanylate cyclase and are haptenic as evidenced by their capacity to raise an antitoxin response in animals immunized with the toxin coupled to a large molecular weight carrier.
The large molecular weight, antigenic heat-labile toxin (LT) has been purified to homogenicity. Its subunit structure has been characterized as (1) five B-subunits that attach the holotoxin (complete toxin) to specific GM.sub.1 ganglioside receptors on the mucosal surface, and (2) a single A-subunit that stimulates intracellular adenylate cyclase activity, thus evoking fluid and electrolyte secretion.
Immunization with either the biologic LT, the biologic LT B-subunit or the biologic ST toxin induces an antitoxin response in experimental animals that protects against homologous and heterlogous serotypes of strains that produce the specific toxin used for immunization. Thus, immunization with LT whole toxin or its B-subunit yields protection against viable heterlogous strains that produce this toxin alone (LT.sup.+ /ST.sup.-) or together with ST (LT.sup.+ /ST.sup.+), but not against those which make just ST(LT.sup.- /ST.sup.+). Similarly, immunization with biologic ST provides protection against direct challenge with viable heterlogous ST-producing strains ((LT.sup.- /ST.sup.+ and LT.sup.+ /ST.sup.+), but not against LT-producing strains.
However, the biologic toxins or the B-subunit are not suitable for immunization when given alone in view of their toxicity, their failure to provide protection against strains that produce the other toxin form, and the fact that the large molecular weight carriers that have been used to render the haptenic biologic ST molecule immunogenic are typically unsuitable for human use. Therefore, the most practical approach for the prevention of ETEC-induced diarrhea would be an immunization program that provides protection against heterologous ETEC serotypes that produce either or both of the LT or ST enterotoxins and does not use an irrelevant carrier molecule.
U.S. Pat. No. 4,411,888 to Klipstein and Klipstein et al., J. Infec. Dis. 147:318-326 (1983) report, inter alia, the development of a vaccine made by conjugating natural or synthetic ST toxin to the biologic B-subunit of LT toxin (LTB) by means of the carbodiimide reaction. As a result of that reaction, synthetic ST acquires immunogenicity when coupled to the large molecular weight LTB carrier, the LTB maintains most of its immunogenicity, while both cross-linked materials lose most of their toxic (secretion-inducing) properties. Rats immunized with the vaccine so produced were reported to be strongly protected against challenge with either LT or biologic ST, as well as against challeange with viable ETEC strains that produce those toxins.
The LTB preparation used for that vaccine was obtained via recombinant methods. That method of preparation, while producing a useful result, is relatively expensive since the resulting recombinant LTB must be purified extensively to remove toxic materials before it can be formulated into a useful vaccinating agent in combination with ST. Recombinant LTB must also be coupled to ST with a covalent linking agent such as soluble carbodiimide, gluteraldehyde, or dimethylsuberimidate, which may not be acceptable in the final formulation for human use. Finally, the size of LTB may necessitate the use of an excess of ST in the preparation of the LT/ST immunogen formulation, which increases costs accordingly.
Richard A. Lerner and co-workers, have shown that a synthetic polypeptide whose amino acid residue sequence corresponds to that of a portion of a protein may be used to elicit antibodies that recognize the intact protein. See Sutcliffe et al. Science 219:660-666 (1984), for a review. Synthetic polypeptides designed following the initial work of Lerner et al. have been successfully used for the preparation of the synthetic ST molecule described before.
The nucleotide sequences coding for the LTB proteins from human- and porcine-infecting (LT.sub.h and LT.sub.p, respectively) E. coli have been determined. Translation of the nucleotide sequences into amino acid sequences leads to proteins whose largest form could contain 124 amino acid residues. See Dallas and Falkow, Nature 288:499-501 (1980) and Yamamoto et al., J. Bacteriol. 155:728-733 (1983).
The 124 residue protein includes 21 amino acid residues at the amino-terminus in what is sometimes referred to as a "signal peptide". Reported amino acid residue position numbers of LTB in the literature consequently may differ by 21 positions, depending upon whether the authors whose work is reported include or exclude the 21 residue signal peptide in their position numbering nomenclature.